IL-2R signaling is essential for functional maturation of regulatory T cells during thymic development

Abstract

CD4(+) Foxp3(+) regulatory T cells (Tregs) are an independent cell lineage, and their developmental progression during thymic development depends on IL-2R signaling. However, the role of IL-2R signaling during thymic Treg development remains only partially understood. The current study assessed the contribution of IL-2 to the expansion and functional programming of developing Tregs. In the absence of IL-2Rβ signaling, predominantly CD4(+) CD25(-) Foxp3(lo) T cells were found, and these cells exhibited somewhat lower expression of the proliferative marker Ki67. These immature Tregs, which represent products of failed development, were also found in normal mice and were characterized by markedly lower expression of several Treg functional molecules. Therefore, IL-2R is required for the progression, functional programming, and expansion of Tregs during thymic development. An IL-2R-signaling mutant that lowers STAT5 activation readily supported Treg functional programming, but Treg proliferation remained somewhat impaired. The requirement for IL-2 during thymic Treg expansion was best illustrated in mixed chimeras where the Tregs with mutant IL-2Rs were forced to compete with wild-type Tregs during their development. Tregs with impaired IL-2R signaling were more prevalent in the thymus than spleen in these competitive experiments. The general effectiveness of mutant IL-2Rs to support thymic Treg development is partially accounted for by a heightened capacity of thymic Tregs to respond to IL-2. Overall, our data support a model in which limiting IL-2R signaling is amplified by thymic Tregs to readily support their development and functional programming, whereas these same conditions are not sufficient to support peripheral Treg homeostasis.

FIGURE 1. Thymic Treg compartment in the absence of IL-2Rβ signaling

(A) Distribution of DN, DP, SP CD4, SP CD8 thymocytes (% positive cells are shown in each quadrant) and (B) total thymic cellularity for the indicated mice. (C) Gating strategy and (D) the frequency of Foxp3⁺ cells in SP CD4 T cells in the thymus of the indicated mice. Data (B, D) are means ± SD from 5-8 mice/group.

FIGURE 2. IL-2 in the regulation of Treg proliferation and Bcl-2 expression

(A) Representative histograms and (B) expression of Bcl-2 and Ki67 in thymic Tregs from the WT, 2Rβ^-/- mice and the host and donor compartment of “cured” 2Rβ^-/- mice. The MFI for Bcl-2 is based on the total Tregs in the respective histogram. Data are means ± SD from 5-8 mice/group.

FIGURE 3. IL-2 regulates expression of Foxp3, CD25 and CD103 in thymic Tregs

(A) Representative dot plots and MFI of Foxp3 and percent of CD25 expression by the indicated Treg populations. (B) Representative histograms and percent of CD103 expression from the indicated thymic Tregs. Data are means ± SD from 3-8 mice/group.

FIGURE 4. IL-2 regulates the thymic Treg functional program

Representative histograms and MFI or percentages of CD39, CD73 and CTLA4 in thymic Tregs from WT, 2Rβ^-/-, and the host and donor compartment of “cured” 2Rβ^-/- mice. Data are means ± SD from 3-8 mice/group.

FIGURE 5. Properties of CD25^- vs. CD25⁺ and Foxp3^lo vs. Foxp3^hi thymic Tregs in normal mice

Representative dots plots (left) for CD25^- vs. CD25⁺ (A) or Foxp3^lo vs. Foxp3^hi (B) by thymic Tregs. Expression (right) of the indicated markers for CD25^- and CD25⁺ (A) or Foxp3^lo and Foxp3^hi (B) Tregs. Data are means ± SD from 5-6 mice/group.

FIGURE 6. Thymic maturation of Treg subpopulations in the presence and absence of IL-2R signaling

Representative dot plots (left) for the indicated thymic populations from WT and 2Rβ^-/- mice and histograms (middle) for CD24 and CTLA4 expression by the cells within the indicated gated regions of the dot plots. The percent of cells within the P1-3 gates for CD24 and CTLA4 are shown to the right. Data are means ± SD from 4-5 mice/group.

FIGURE 7. Treg developmental progression and maturation in the neonatal thymus of WT mice

In each panel, thymocytes were always first gated on CD4⁺ CD8^- thymocytes. The number (A) and expression of CD25 (B) by Foxp3⁺ cells from Foxp3/RFP reporter mice of the indicated age. Data are means ± SD from 4-6 mice/group, except day 1 (n=2). (C) Comparison of CD25 expression by Foxp3⁺ T cells from mice of the indicated age after intracellular staining for Foxp3 protein. (D) Representation of the CD4⁺ CD25^- Foxp3^- population from Foxp3/RFP reporter mice of the indicated age. (E-F) Expression of CD39 and CD103 by Foxp3⁺ cells from Foxp3/RFP reporter mice of the indicated age. Data (C-F) are means ± SD from 3-6 mice/group.

FIGURE 8. Characterization of thymic Tregs with sub-optimal IL-2Rβ signaling

(A) Representative dot plots and (B) expression of the indicated molecules by Foxp3^lo vs. Foxp3^hi thymic Tregs from the indicated mice. Data are means ± SD from 3-5 mice/group. Statistically significant differences are shown for comparisons between WT and Y3.

FIGURE 9. pSTAT5 activity of thymic Treg cells

(A). Representative histograms and (B) percent and MFI of pSTAT5 expression by the indicated mice. Data are means ± range from 2 mice/group. (C) Percent and (D) relative MFI (thymus:spleen) of pSTAT5 expression directly ex vivo by WT B6 and Y3 thymic and splenic Tregs. Data are means ± SD from 5 mice.

FIGURE 10. Effectiveness of low IL-2R signaling for Treg thymic development and peripheral homeostasis in competitive chimeric mice

(A) Experimental design for the mixed bone marrow chimeras. (B) Representative histograms to follow donor-derived SP CD4⁺ Foxp3^- T cells and CD4⁺ Foxp3⁺ Tregs in the thymus and spleen. (C) The relative development of Tregs in the thymus and spleen of chimeric mice. The x-axis shows the indicated CD45.2 donor bone marrow, the ratio (CD45.2:CD45.1) after mixture with CD45-1⁺ WT B6 bone marrow, and the irradiated recipient. The y axis represents Efficiency of Treg reconstitution of the indicated CD45.2 Tregs and was calculated by the formula: Efficiency= (% Treg_CD45.2/% Treg_CD45.1)/(% SP CD4⁺ T conventional_CD45.2/% SP CD4⁺ T conventional_CD45.1). Values >1 or <1 for Efficiency represent over or under-representation of the CD45.2⁺ donor-derived Treg population, respectively. (D) The expression of CD25 by CD45.2 Tregs from the indicated chimeras. The expression of CD25 by the CD45.2⁺ Tregs was normalized to the CD45.1⁺ Tregs within the same mouse, i.e. Relative CD25 MFI= MFI_CD45.2/MFI_CD45.1. Data (C,D) are means ± SD from 3-5 mice/group.